Alzheimer?s focused administrative supplement for R01 EY028219 [Notice Number: NOT-AG-18-039] Title: Glial mechanisms of AD in visual cortex Abstract: Alzheimer?s disease (AD) is a devastating neurodegenerative disorder characterized by progressive cognitive decline, memory loss, behavioral impairment, and death. Pathological hallmarks of AD, such as intracellular neurofibrillary tangles and extracellular amyloid-beta (A?) aggregations, represent abnormal neural homeostasis; however, the pathogenesis of these hallmarks and the circuit dysfunction that leads to these abnormalities remain poorly understood. Moreover, effective therapeutic targets for AD and related disorders continue to be scarce despite substantial research efforts. Previous studies of AD pathogenesis have identified astrocytes and microglia as critical players in disease progression, suggesting that glial mechanisms may be valuable therapeutic targets. Recent evidence has shown that gamma oscillations (at ~40Hz) in visual cortex network activity driven by parvalbumin (PV) expressing interneurons alter microglial responses to A? deposition in a mouse model of AD, with a corresponding increase in microglial phagocytic gene expression. The mechanisms by which visual stimuli modulate glial signaling and improve AD pathogenesis are poorly understood. Through our work in EY028219, we have directly measured the impact of visual stimuli on astrocyte calcium transients and demonstrated that they reflect PV (and other neuron) activity via glutamate and GABA transporters. Ongoing work in our lab has further demonstrated that the neuromodulator norepinephrine (NE), released from the locus coeruleus (LC), modulates both astrocytic signaling and microglia dynamics, suggesting that NE is a key factor in maintaining glial functions. In several studies of AD, the degeneration of the LC and subsequent alteration in brain-wide NE release has been linked to disease onset and progression, providing the intriguing possibility that dysregulation of glial functions may be causally linked to decreased NE signaling. Furthermore, NE enhances the activity of PV neurons directly, and influences network activity in visual cortex via diverse cell-specific receptor classes; LC-NE activation itself is strongly modulated by sensory stimuli. In this supplemental application, we propose to determine the effect of visual gamma frequency entrainment and of NE release on neuro-glial signaling (Aim 1), and determine the effect of NE release on gamma-frequency-dependent neuro-glial dynamics in a mouse AD model (Aim 2). In Aim 1a, we will use in vivo 2-photon microscopy to image microglial dynamics and astrocytic Ca2+ responses in visual cortex during 40Hz visual stimulation. We will also assess endogenous NE release during 40Hz visual stimulation by recording Ca2+ in LC-NE axons. In Aim 1b, we will use pharmacological and optogenetic methods to alter LC-NE release during visual stimulation and characterize the effects on microglia and astrocytes. In aim 2a, we will use the 5xFAD mouse model as previously described to study the effect of NE release and 40Hz visual stimulation on neuro-glial dynamics in the context of AD. These mice recapitulate several AD-related phenotypes, such as severe A? pathology beginning around 1.5 months of age, gliosis developing in parallel with plaques, and synapse degeneration. We will image A?-plaques using methoxy-X04 together with astrocyte or microglial imaging, as demonstrated by our preliminary data. In aim 2b, we will modulate NE activity in 5xFAD mice by using optogenetic and pharmacological approaches described in Aim 1 and evaluate the effect of NE stimulation or suppression alone or with 40Hz visual stimulation in the modulation of glial responses and A?-plaque burden. These experiments all lie within the scope of the active award. The experiments are focused on AD, and we expect they will stimulate additional work in our lab, and in collaboration, that will contribute to progress on AD. Specifically, these experiments will provide in vivo evidence to characterize the effects of gamma frequency neuronal stimulation and NE on glial dynamics in the healthy and AD mouse brain. The results from the proposed studies will shed new light on the cellular mechanisms contributing to neuro-glial interactions and homeostatic disruption, and may provide new therapeutic targets for the treatment of AD and associated age-related dementias.